Sir2 (Silent Information Regulator-2) proteins function in aging and lifespan regulation in multiple model organisms. Our long-term goal is to understand the molecular mechanisms that underlie human aging and age-associated pathologies, and the role of human Sir2 factors in attenuating these processes. Preliminary work indicates that one mammalian Sir2 protein, SIRT6, suppresses genomic instability and attenuates the onset of several age-associated pathologies. However, the molecular mechanisms of SIRT6 function are not known. Thus, a systematic characterization of the basic molecular mechanisms through which SIRT6 functions in human cells should be instrumental for elucidating fundamental biological processes that impact on human health in aging. Here, the overall hypothesis to be investigated is that SIRT6 exerts its effects on aging-associated cellular processes and genome maintenance via novel functions at chromatin. A series of biochemical, cellular, and global proteomic and genomic analyses will be carried out to define the molecular functions of SIRT6 in human cells. Three Specific Aims are proposed: 1. To elucidate the molecular mechanisms by which SIRT6 functions in chromatin regulation. Post-translational modifications of histones at chromatin play important roles in gene expression programs and DNA damage responses. Preliminary results indicate that SIRT6 is tightly associated with, and may catalyze modifications of, histones at chromatin. We will define the enzymatic activity of SIRT6 at chromatin and the effects of altered SIRT6 levels on chromatin structure and functional states. 2. To elucidate the role of the human SIRT6 protein in genome stabilization and DNA repair. SIRT6-deficient cells show increased genomic instability and sensitivity to oxidative and alkylating DNA lesions that are proposed to contribute to aging. A series of functional and biochemical experiments are proposed to elucidate the molecular mechanisms by which SIRT6 exerts its effects on genome maintenance. 3. To elucidate novel SIRT6 regulated molecular pathways. Proteomic and biochemical approaches will be taken to isolate and characterize SIRT6 substrates, protein binding partners, and macromolecular complexes. In addition, genome wide approaches will be employed to identify SIRT6-regulated genes and SIRT6 binding sites at chromatin.